Poster S16 - Engineering Targeted Nanoparticles to Combat Triple-Negative Breast Cancer

Introduction: Triple-negative breast cancer (TNBC) is an aggressive and highly metastatic form of breast cancer that lacks effective treatment options. Only recently have targeted therapies for TNBC begun to enter the market, with the antibody-drug conjugate (ADC) Trodelvy being approved by the Food and Drug Administration in 2020. Trodelvy has seen success in treating patients with TNBC because it targets an extracellular receptor, Trop2, which is overexpressed in tumor cells, for delivery of a small molecule drug, SN38. Although more time is needed to understand the full clinical performance of Trodelvy, current limitations include its short half-life ( < 1 day) and significant off-target effects including life-threatening neutropenia and severe diarrhea. While SN38 alone holds promise as an anticancer drug, its poor aqueous solubility and high systemic toxicity necessitate the use of a carrier. We hypothesize that the limitations of Trodelvy can be overcome by loading SN38 in targeted nanocarriers. To test this hypothesis, SN38 is encapsulated in poly (lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) that are either: (i) coated with Trop2 antibodies (AbNP), or (ii) wrapped with TNBC cell-derived membranes (MWNP). Previous work in our lab has shown that antibody-NP conjugates and TNBC cell membrane wrapped NPs exhibit greater binding to targeted cells because they can engage multiple receptors simultaneously via multivalent binding and homotypic targeting, respectively. Here, we report the synthesis and characterization of each SN38 nanocarrier. Ongoing work is comparing each nano-carrier head-to-head with the ADC to reveal which formulation provides the most efficient tumor targeting and most potent therapeutic effects.

Materials and

Methods: SN38-loaded PLGA NPs were synthesized based on our published methods. To synthesize AbNPs, we used maleimide-functionalized PLGA for SN38 encapsulation and anti-Trop-2 antibody conjugation. The anti-Trop-2 antibody was cleaved with IdeZ protease and reduced using DTT to form anti-Trop-2-SH. Anti-Trop2-SH was incubated overnight with mal-PLGA NPs to complete click chemistry reaction to form antibody-conjugated NPs (AbNPs), as described in Fig. 1. To synthesize MWNPs, 4T1 cells were lysed and mechanically homogenized, followed by ultracentrifugation to extract membranes. The membranes were co-extruded with NPs to form membrane-wrapped NPs (MWNPs) as described in Fig. 2. To characterize the AbNPs, and MWNPs, we measured their hydrodynamic size by nanoparticle tracking analysis, zeta potential by dynamic light scattering, and structure by transmission electron microscopy (TEM). SN38 loading efficiency was analyzed by measuring absorbance (390 nm) of the non-encapsulated/released drug. Anti-trop-2 antibody conjugation was quantified by ELISA. TNBC cell membrane coating was confirmed by measuring specific membrane proteins with Western blot. Moving forward, the TNBC cell targeting of each formulation will be tested in vitro by exposing 4T1 cells to Cy5-tagged AbNPs, MWNPs, or free Cy5-tagged Trop-2 antibodies and measuring cellular uptake by flow cytometry and confocal microscopy.

Results, Conclusions, and Discussions: Fig. 3 and Fig. 4 display the characterization of AbNPs and MWNPs for SN38 delivery, respectively. As seen in Fig. 3A, the NP diameter increases by ~10 nm after antibody conjugation compared to bare NPs. Likewise, Fig. 3B shows that the zeta potential shifts from -27 mV for bare NPs to -33 mV for AbNPs, indicating successful antibody attachment. Per TEM images (Fig. 3C), the NPs are spherical and uniform in size. Importantly, there is minimal loss ( < 20%) of SN38 cargo during the antibody conjugation process, with Bare NPs and AbNPs having ~177 and ~146 µg SN38/mg PLGA, respectively (Fig. 3D). ELISA results indicate the AbNPs are coated with ~236 antibodies per NP (Fig. 3D). Characterization for MWNPs is shown in Fig. 4. The hydrodynamic size of the NPs increases by ~20 nm after membrane coating (Fig. 4A) and the zeta potential shifts from -26 mV for Bare NPs to -31 mV for MWNPs, which mimics the -33 mV charge of the cancer cell membrane vesicles (Fig. 4B). In TEM images, the membrane layer surrounding the NPs is clearly visible (Fig. 4C), confirming successful wrapping. Finally, Fig. 4D shows the SN38 loading pre- and post-membrane wrapping of the NPs. Compared to Bare NPs, wrapped NPs have ~25% less SN38 cargo, which may be due to loss of surface-adsorbed drug during the extrusion process used to form MWNPs.

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